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This study used Silibinin as an additive to conduct fermentation experiments, wherein its effects on rumen gas production, fermentation, metabolites, and microbiome were analyzed in vitro . The silibinin inclusion level were 0 g/L (control group), 0.075 g/L, 0.15 g/L, 0.30 g/L, and 0.60 g/L (experimental group). Fermentation parameters, total gas production, carbon dioxide (CO 2 ), methane (CH 4 ), hydrogen (H 2 ), and their percentages were determined. Further analysis of the rumen microbiome's relative abundance and α/β diversity was performed on the Illumina NovaSeq sequencing platform. Qualitative and quantitative metabolomics analyses were performed to analyze the differential metabolites and metabolic pathways based on non-targeted metabolomics. The result indicated that with an increasing dose of silibinin, there was a linear reduction in total gas production, CO 2 , CH 4 , H 2 and their respective percentages, and the acetic acid to propionic acid ratio. Concurrent with a linear increase in pH, when silibinin was added at 0.15 g/L and above, the total volatile fatty acid concentration decreased, the acetic acid molar ratio decreased, the propionic acid molar ratio increased, and dry matter digestibility decreased. At the same time, the relative abundance of Prevotella , Isotricha , Ophryoscolex , unclassified_Rotifera , Methanosphaera , Orpinomyces , and Neocallimastix in the rumen decreased after adding 0.60 g/L of silibinin. Simultaneously, the relative abundance of Succiniclasticum , NK4A214_group , Candidatus_Saccharimonas , and unclassified_Lachnospiraceae increased, altering the rumen species composition, community, and structure. Furthermore, it upregulated the ruminal metabolites, such as 2-Phenylacetamide, Phlorizin, Dalspinin, N6-(1,2-Dicarboxyethyl)-AMP, 5,6,7,8-Tetrahydromethanopterin, Flavin mononucleotide adenine dinucleotide reduced form (FMNH), Pyridoxine 5'-phosphate, Silibinin, and Beta-D-Fructose 6-phosphate, affecting phenylalanine metabolism, flavonoid biosynthesis, and folate biosynthesis pathways. In summary, adding silibinin can alter the rumen fermentation parameters and mitigate enteric methane production by regulating rumen microbiota and metabolites, which is important for developing novel rumen methane inhibitors., Competing Interests: YS was employed by Beijing Sunlon Livestock Development Company Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Shen, Ma, Li, Lambo, Dai, Shen, Qu and Zhang.)